Detection of dysplastic or neoplastic cells using anti-MCM2 antibodies

ABSTRACT

Determination of cellular growth abnormality, particularly cancerous abnormality, by detection of target polypeptides or encoding mRNA, where the target polypeptides are members of the preinitiation complex of DNA replication in tissue, cells or fluid. Target polypeptides include CDC6, MCM2, MCM3, MCM4, MCM5, MCM6 and MCM7. Test samples include tissue of the cervix (both biopsy and smear samples), breast, colon, lung, bladder, skin, larynx, oesophagus, bronchus, lymph nodes and urinary tract (both biopsy and cytology smear samples), in determination of cancerous and precancerous cellular growth abnormality, and cells spun from urine, blood and serum, in determination of haematological malignancies and evidence of metastatic sarcoma and carcinoma.

This is a divisional application of Ser. No. 09/175,947 (allowed) whichwas filed Oct. 21, 1998 now U.S. Pat. No. 6,303,323, this applicationclaims the benefit claims the benefit of U.S. Provisional ApplicationNos. 60/071,245, 60/086,885 and 60/095,966, filed 22 Dec. 1997; 27 May1998; and 10 Aug. 1998, respectively, the entire contents of which arehereby incoroorated by reference in the application.

The present invention relates to assessment of cells in a sample oftissue, cells or fluid with a view to detecting cellular growthabnormality, particularly potentially (or actually) cancerous cells.Aspects of the present invention are particularly useful in screeningsamples such as cervical smears from women to detect those whosecervical cells are abnormal. The invention is also applicable toassessment of cells in other tissue samples, including breast, asdemonstrated experimentally herein. Samples found to be abnormal may beexamined in more detail and the condition of cells in the tissueinvestigated further. Identification of a malignant or pre-malignantcondition may be followed by appropriate treatment following moreextensive diagnostic procedures.

The present invention is based on the surprising discovery that specificbinding molecules directed against particular proteins of thepreinitiation complex of DNA replication can be used to detect abnormalcells. Especially useful in the present invention are binding moleculesdirected against Cdc6. Also especially useful are binding moleculesdirected against MCM proteins, particularly MCM5. Experimental evidenceincluded herein shows that specific binding molecules directed againstCdc6, and also those against MCM2, MCM3, MCM4, MCM5, MCM6 or MCM7 aremuch more effective in marking cellular growth abnormality in tissuesamples than antibodies against PCNA and Ki67. A priori one would haveexpected Cdc6 and the MCM's to give similar results as Ki67 and PCNA,since all these proteins can be considered “proliferation markers”. Oncervical samples subject to antigen retrieval (pressure cooking orautoclaving), experimental results below show that in fact resultsobtained are similar for all these, but there is clear difference oncervical smears and frozen samples. Such samples, of primary interestfor screening purposes, are not robust enough to be subject to pressurecooking. Of particular interest in the context of screening are the verystrong and clear results obtained with assessment of cervical samplesusing anti-Cdc6 or anti-MCM binding molecules, showing high-levelstaining of abnormal cells, and full-thickness staining in LSIL samples.This indicates usefulness in assessment of smear samples taken from thecervical epithelial surface—and indeed this is verified experimentallyherein. Full thickness staining is also seen for HSIL samples.

Experimental assessment of abnormality in breast tissue, urine, bloodand serum confirms generality of aspects of the present invention.Further evidence is provided by the use of the same antibodies indetection of the presence of dysplastic or neoplastic cells in bodyfluids by biochemical methods that can be automated. Examplesdemonstrated herein include detection of bladder cancer by analysis ofurine and detection of both leukaemia and lymphoma by analysis of blood.A suitable method for such analysis is Dissociation Enhanced LanthanideFluorescence Immunoassay, “DELFIA”. Also included is demonstration ofdetection of sarcoma and carcinoma cases by DELFIA on blood.

The cervical epithelium is essentially composed of two distinct celltypes: the squamous epithelium and the columnar epithelium, each ofwhich is located in an anatomically distinct region of the tissue. Thesquamous epithelium is located at the exterior aspect (the ectocervix)of the cervical opening (os), while the columnar epithelium extends intothe endocervical canal (the endocervix). These two distinct epithelialcell types come into contact in the vicinity of the cervical os, at thesquamo-columnar junction. The squamo-columnar junction is of clinicalimportance as it is the region where the majority of malignancies arise.For diagnostic validity, a cervical smear sample should include cellsfrom this region. In order to ensure that this has been achieved, asmear should contain columnar as well as squamous epithelial cells.

Most cervical tumours arise at the squamo-columnar junction from thesquamous epithelium, which is a multilayered dynamic stem cell systemunder constant renewal. The stem cell compartment itself is locatedadjacent to the basement membrane within the basal cell layer. Stem celldivision gives rise to parabasal, intermediate, and superficial cellderivatives. These are conventionally defined in terms of both theircharacteristic morphology and location within the squamous epithelium.The transition from basal cells located in the deepest layer of thesquamous epithelium, to superficial cells at its surface is associatedwith progressive differentiation and a loss of proliferation untilsuperficial squamous epithelial cells at the cervical surface areterminally differentiated.

In dysplasia, there is increased cellular proliferation with a reductionin differentiation of cells as they progress through the squamousepithelium. Typically, for convenience in the first instance, cervicalscreening involves assessment of smears taken from the surface of theepithelium, the cytopathologist looking for abnormalities at the surfacerepresentative of reduced differentiation as a result of dysplasia.

At the late foetal stage, during adolescence and in pregnancy columnarepithelium is replaced at the junction by squamous epithelium by aprocess of metaplasia. Metaplastic squamous cells which replace columnarcells are particularly vulnerable to carcinogens. Normal metaplasiashould not be confused with abnormal dysplasia within the squamousepithelium, and it can be important in screening contexts to be able todistinguish between metaplastic and dysplastic cells.

Despite an intensive and expensive national screening programme,carcinoma of the cervix is the eighth most common malignancy of women inthe UK and the most common malignancy in women under 35 years of age(Cancer Research Campaign, Cancer of the cervix uteri. 1994, CRC:London). In the developing world it is the most common malignancy andthe leading cause of death in women between the ages of 35–45 years,with an estimated 437,000 new cases each year (Cancer Research Campaign,Cancer—world perspectives,. 1995, CRC: London).

The majority of cases represent squamous cell carcinoma (SCC) and arestrongly associated with infection with ‘high-risk’ types of humanpapillomavirus, such as 16, 18 and 31 (Park, et al. Cancer, 1995, 76 (10Suppl.): p. 1902–13). Cervical carcinoma is amenable to prevention bypopulation screening, as it evolves through well-defined non-invasive‘intraepithelial’ stages (Wright, et al. Precancerous lesions of thecervix, in Blaustein's pathology of the female genital tract. R. J.Kurman, Editor. 1994, Springer-Verlag: New York. p. 229–78). Squamousintraepithelial abnormalities may be classified using 3 tier (CIN) or 2tier (Bethesda) systems. Different histological abnormalities broadlycorrelate with the type of infecting HPV and with the DNA ploidy,clonality and natural history of the lesion. As classified by theBethesda system, low grade squamous intra-epithelial lesions (LSIL),corresponding to CIN1 and cervical HPV infection (HPVI) generallyrepresent productive HPV infections, with a relatively low risk ofprogression to invasive disease (Wright and Kurman. A critical review ofthe morphological classification systems of preinvasive lesions of thecervix: the scientific basis for shifting the paradigm, inPapillomavirus reviews: current research on papillomaviruses, C. Lacey,Editor. 1996, Leeds University Press: Leeds). High-grade squamousintra-epithelial lesions (HSIL), corresponding to CIN2 and CIN3, show ahigher risk of progression than CIN1 (LSIL) though both are viewed asrepresenting a potential precursor of malignancy. Although it ispossible to estimate the approximate risk of malignancy for eachcategory of intra-epithelial lesion, it is currently not possible todetermine the approximate likelihood of progression for an individualcase.

In 1943, Papanicolau and Trout introduced the Pap smear test to detectprecursors of cervical cancer in women. This is a cytological screeningtest and has probably proved to be the most successful public healthmeasure introduced for the prevention of cancer. Mass screeningprogrammes, in which women have cervical smear tests at least once everythree to five years, have proven highly effective in some countries inreducing cervical cancer mortality and morbidity rates. In BritishColumbia and Finland for example, organised screening has reducedmortality rates for cervical cancer by 70%. If detected early, cervicalcancer is easily treated.

In spite of these achievements, the reality of the situation world-wideis depressing. Of the hundreds of thousands of women who developcervical cancer annually, more than 50% will die of the disease. Seventyfive per cent of all those women will be in the developing world, wherebecause of financial constraints, mass screening programmes usingavailable methodologies are non-viable. Even in many developedcountries, the decline of the disease in the past decade has beeninsignificant, while the impact of cytological screening has been farless than expected. In addition, experts observe a substantialproportion of cases of invasive cervical cancer in patients who areregularly screened, particularly young women.

The major reasons why cytological screening sometimes fails to detectcervical cancer are the large intervals between tests and also the highnumber of false negative results (10–30%) (Pap Cytology screening: Mostof the benefits reaped? WHO and EUROGIN release a report on cervicalcancer control. Press Release WHO/25, March 1997).

The high number of false negative results reflects the fact thatinterpretation of Pap smears is one of the most difficult ofmorphological exercises. The results of a Pap smear are harder tointerpret than those of fine needle aspiration, body fluid cytologicaltesting or biopsies because of the complexity and variability of themixed cell population placed on the smear and the wide range ofinflammatory and reparative processes that occur in the cervix. Thereare also cyclical changes in the cellular population, pregnancy inducedalterations and alterations that occur in the postmenopausal period.Because gynaecological cytology is so difficult, the training periodsfor cytotechnologists are long; they require an educated student andhigh degree of discipline and pattern recognition skills. Even aftercompleting an adequate training programme, cytotechnologists requireseveral years of practical experience before they can make consistentlyaccurate judgements as to whether a Pap smear result is normal orabnormal. Similarly, although pathologists may be trained to interprethistological sections, they require specialised additional training incytopathology to possess adequate skills to organise and supervise thecytology laboratory and to make appropriate diagnoses concerningabnormal smears.

The two major problems associated with Pap screening programmes are anapparently unavoidable false negative rate (10–30%) and the relativelyhigh cost of screening. Therefore, alternative approaches are now beingconsidered to cervical screening. The two most commonly discussedproposals are to use HPV DNA testing and typing as a primary screeningmodality or as a supplement to Pap smears and to use instruments thatcan automatically screen conventionally taken Pap smears, thus reducingthe need for the relatively highly paid cytotechnologists andcytopathologists (Richart, Cancer Supplement, 1995, 76(10): 1919–1927;Birdsong, Human Pathology, 1996, 27(5): 468–481).

The former method remains problematical. There are problems ofsensitivity and positive predictive value using in-situ methods for HPV.The use of PCR for HPV DNA detection produced such high rates of HPVinfection in the general population that HPV DNA testing is thought tobe of questionable use for clinical screening.

The second approach involves automation. A number of companies arecurrently developing and marketing automated screening instruments. Ingeneral such instruments use a high resolution video scanner to captureimages, which are then digitalised and analysed with a series ofalgorithms, and the data are then passed through an interference networkthrough which the machine has been trained to distinguish between normaland abnormal cellular components. It is hoped that with further softwareand hardware development, automated screening can be considered forprimary screening, though at the moment no devices have been approvedfor the pre-screening or independent screening of Pap smears by the USFDA. That companies are prepared to invest so heavily in such anexpensive and complex approach in attempting to overcome problems withconventional PAP smear testing illustrates the severity of the problemsand the heart-felt need for a solution.

Assessment of cell proliferation markers has not previously provided anysuch solution and experts in the field have been sceptical thatproliferation markers will provide useful clinical information (Hall andCoates, Histopathology, 1995, 26: 105–112). There is a belief thatmeasuring parameters of cell proliferation will provide objectiveinformation about tumours, but despite numerous studies there is littledirect evidence that the use of cell proliferation markers such as PCNA,Ki67 etc. are really an improvement on conventional histologicalassessment optimally employed. Few studies have even addressed thecritical issue of the relative value of proliferation markers comparedto the standard histopathological grading and staging.

Attempts to use immunocytochemical or immunofluorescent staining withautomated cervical screening have been limited by non-specific stainingof normal cells. For example, epithelial membrane antigen (EMA) has beenshown to stain neoplastic cells from cervices with CIN, but staining ofsome metaplastic cells from normal cervices was also reported.Therefore, although technology for measuring immunchistological stainingis available, none of the automated screening machines that are on themarket or in advanced development use immunohistochemistry at this time.

The most widely studied markers of proliferation are Ki67, a protein ofunknown function and PCNA (proliferating cell nuclear antigen) (Yu andFilipe, Histochemical Journal, 1993, 25: 843–853). PCNA is involved inthe elongation of DNA replication and in the mechanism of DNA repair.Therefore it is present during actual DNA synthesis by replication orrepair.

The present inventors have studied proteins involved in the earlierinitiation stage of DNA replication. These are Cdc6 and proteins of theMCM2–7 family (MCM2, MCM3, MCM4, MCM5, MCM6 and MCM7). Williams et al(1997) (Proc. Natl. Acad. Sci. USA, 1997, 94: 142–147) reported thathuman HeLa Cells in culture express Cdc6 throughout proliferating cellcycles, but that WI38 human diploid fibroblasts stop expressing Cdc6when made quiescent by serum starvation. It is shown herein that theseobservations extend to other cell lines and other species. MCMs arepresent in G1 phase nuclei (before DNA synthesis) and are progressivelydisplaced from chromatin into the soluble nucleoplasm during DNAsynthesis. It is shown herein that they too are absent from chromatinduring quiescence. It is also demonstrated herein that MCM5 is absentfrom differentiated cells of the uterine cervix and breast.

From these background facts, MCMs or Cdc6 antisera would be expected toresemble the distributions of PCNA or Ki67. Further evidence for thisexpectation comes from Hiraiwa et al (Int. J. Cancer, 1997, 74: 180–184)who found similar immuno-staining patterns for PCNA and MCM7 (hCDC47) inseveral human tissues and three types of human tumour.

Surprisingly, however, the present inventors have found dramaticdifferences in the potential diagnostic value of MCMs and Cdc6 ascompared with PCNA and Ki67.

The inventors have tested antisera raised against human MCM protein andhuman Cdc6 for cervical cytology. They have studied sections of normaland diseased human uterine cervix and cervical smears. They havecompared the results with those obtained using PCNA and Ki67. Cdc6antibodies or MCM (e.g. MCM5) antibodies detect LSIL (HPVI/CIN 1)lesions in the cervix more effectively than antibodies against PCNA orKi67. Furthermore, essentially all cells of LSIL (HPVI/CIN 1) or HSIL(CIN 2/3) lesions are stained. This is in contrast to staining by otherproliferation markers such as PCNA. It indicates that specific bindingmolecules directed to proteins of the preinitiation complex of DNAreplication, particularly Cdc6 or MCM proteins (such as MCM5 but alsoexemplified herein for MCM2, MCM3, MCM4, MCM6 and MCM7) have exceptionaldiagnostic value for early detection of atypical or neoplastic cells. Oncervical samples subject to antigen retrieval (pressure cooking orautoclaving), which samples are formalin fixed and paraffin embedded,anti-Cdc6 and anti-MCM antibodies give similar patterns of staining tothose obtained with PCNA, but the superior results on smears, fresh andfrozen samples are clear.

Thus, the present invention generally relates in various aspects tomethods and means for detecting a particular target polypeptide, or mRNAencoding a target polypeptide, in tissue, fluid or cells of anindividual, usually in a sample removed from the body.

Target polypeptides of the present invention such as Cdc6 and MCMproteins, such as MCM5, may be distinguished from other cellularproliferation markers which are not useful in the present invention bybeing included within the preinitiation complex of DNA replication. Theymay be distinguished by being displaced from chromatin during quiescenceand differentiation. ORC2 (Gavin et al., 1995, Science 270, 1667–1671)for example, which is not a target for use in the present invention, maybe distinguished from proteins such as Cdc6 and MCM5 by remaining boundto chromatin in quiescent cells. Orc2 is not down-regulated in quiescentcells, though other components of the ORC complex, such as Orc1, maybehave differently. Cdc6 is down-regulated rapidly during quiescence anddifferentiation. Cultured cells arrested in G0 for as little as 48 hoursdo not contain any detectable Cdc6 protein. Cdc6 is not detectable incells arrested in vitro for longer periods of time or in differentiatedcells ex vivo. Cells arrested in vitro by serum starvation or contactinhibition lose chromatin-bound MCMs (after a few days), although thetotal level of MCMs in the cells does not decrease appreciably, at leastwithin 14 days. Cells which undergo differentiation in vitro (e.g HL-60cells induced to differentiate with DMSO or TPA) down-regulate MCM3 butnot Orc2 (Musahl, Aussois Meeting on DNA Replication, Aussois, France,June 1997). Differentiated cells from tissues ex vivo do not express MCMproteins such as MCM2 and MCM5. The six MCM proteins MCM2–MCM7 form amultiprotein complex, which splits into two subcomplexes: MCM3 and MCM5dimer; MCM2-4-6-7 tetramer. MCM3 and MCM5 may be displaced fromchromatin during S phase more slowly than MCM2-4-6-7 (Kubota et al.,1997, EMBO J. 16, 3320–3331). MCMs are chromatin-bound in G1, displacedduring S phase and nuclear, but not bound to chromatin in G2. Cdc6behaves similarly in yeast, though in addition to being displaced fromchromatin it is also degraded, protein levels going down dramatically atG1/S transition. Further components of the preinitiation complex of DNAreplication may be included in accordance with the present invention.Examples include human homologues of yeast components, such as Cdc7protein kinase (Chapman and Johnston, Exp. Cell Res., 1989, 180 419–428(yeast), Sao et al., 1997, EMBO J., 16, 4340–4351 (human—down-regulatedin quiescence)), Dbf4, the regulatory subunit of Cdc7 protein kinase(Jackson et al., 1993, Mol. Cell Biol. 13 2899–2908 (yeast), Masai etal., Cold Spring Harbor Meeting on Eukaryotic DNA Replication, Sep. 3–71997 (human)), Cdc14 protein phosphatase (Hogan and Koshland PNAS USA,1992, 89, 3098–3102 (yeast)), Cdc45, which associates with and has asimilar phenotype to MCMs (Zou et al., Mol. Cell. Biol., 1997, 17,553–563 (yeast), Takisawa et al., Cold Spring Harbor Meeting OnEukaryotic DNA Replication, Sep. 3–7 1997 (Xenopus)), MCM10, whichassociates with and has a similar phenotype to MCMs (Merchant et al.,1997, Mol. Cell Biol. 17 3261–3271). Target polypeptides of the presentinvention may variously be said to be any of components of the DNApre-replicative complex, components of replication competent chromatin,involved in restricting DNA replication to once per cell cycle,components of the replication licence, involved in licensing chromatinfor a single round of DNA replication, and assembled at replicationorigins before initiation of DNA replication.

Human Cdc6 amino acid sequence is disclosed in Williams et al., 1997,PNAS USA 94: 142–147, GenBank Acc. No. U77949.

Human MCM2 sequence is disclosed in Todorov et al., 1994, J. Cell Sci.,107, 253–265, GenBank Acc. No. X67334.

Human MCM3 sequence is disclosed in Thommes et al., 1992, Nucl. AcidRes., 20, 1069–1074, GenBank Acc. No. P25205.

Human MCM4 sequence is disclosed in Ishimi et al., 1996, J. Biol. Chem.,271, 24115–24122, GenBank Acc. No. X74794.

Human MCM5 sequence is disclosed in Hu et al., 1993, Nucleic Acids Res.,21, 5289–5293, GenBank Acc. No. X74795.

Human MCM6 sequence is disclosed in Holthoff et al, 1996, Genomics, 37,131–134, GenBank Acc. No. U46838.

Human MCM7 sequence is disclosed in Hu et al., 1993, Nucleic Acids Res.,21, 5289–5293.

According to the one aspect of the present invention there is provided amethod of determining the presence or absence of abnormallyproliferating cells or cellular growth abnormality in a sample from anindividual, the method including contacting a sample with a specificbinding member directed against a target polypeptide, as discussed, anddetermining binding of the specific binding member to the sample.

Another aspect of the present invention provides for a method ofcategorising a tissue as (i) normal or (ii) potentially or actuallypre-cancerous or cancerous, dysplastic or neoplastic, the methodincluding determining binding to a sample of the tissue of a specificbinding member directed against a target polypeptide, as discussed. Thepattern or degree of binding may be compared with that for a knownnormal sample and/or a known abnormal sample.

Human Cdc6 has been cloned independently by the present inventors, asdescribed herein, but the first publication of its cloning was byWilliams et al, whose paper (PNAS USA 94: 142–147, 1997) provides thefull amino acid sequence. As demonstrated experimentally herein,anti-Cdc6 binding molecules are very effective in marking abnormality invarious tissues, especially cervical samples, preferably smears. Thiscompares with no binding to normal cervical tissue in a smear sample.

The amino acid sequence for human MCM5 is disclosed in Hu et al., 1993,Nucleic Acids Res., 21, 5289–5293, GenBank Acc. No. X74795. Experimentalevidence included herein shows that binding molecules directed againstit, like Cdc6, are very effective in marking abnormality in varioustissues, especially cervical samples, preferably smears. Obtaining highaffinity antibodies against MCM5 seems easier than for Cdc6, which mayreflect higher antigenicity.

Further experimental evidence included herein shows that bindingmolecules directed against MCM2, against MCM3, against MCM4, againstMCM6 or against MCM7 are also effective in marking abnormality in tissuesamples such as cervical smears. Anti-MCM5 antibodies have been found togive a stronger staining pattern than anti-MCM2 and anti-MCM7antibodies, both overall and in the number of cells. Anti-CDC6antibodies have been found to give a similar staining pattern toanti-MCM5.

Thus, binding of (e.g.) an anti-Cdc6 or anti-MCM specific binding memberto a sample provides for categorising the tissue from which the sampleis derived as abnormal, potentially or actually pre-cancerous orcancerous, dysplastic or neoplastic. In accordance with present practiceupon obtaining a positive result using the Pap test, apositively-testing individual may be investigated further, for instanceby means of biopsy testing and/or repeat screening. It is quite commonfor pre-cancerous potential not to result in an actually cancerousstate. Six-monthly screening is typically used to follow progression orregression of dysplasia to allow for appropriate and timely therapeuticintervention if required.

If a tissue is categorised as potentially or actually pre-cancerous orcancerous, on the basis of detected abnormality in a tissue sample inaccordance with the present invention, appropriate diagnostic and/orclinical follow-up will be called for.

It is notable that the invention is not limited to detection of cellulargrowth abnormality that is necessarily pre-cancerous or cancerous. Otherdisorders of cellular proliferation may be detected, as is demonstratedby the experimental exemplification included below, including forpsoriasis (see Example 24 below) and inflammatory bowel disease such asulcerative colitis and Crohn's disease (Examples 33 and 34). In additionto being cellular proliferation disorders in their own right,inflammatory bowel diseases may be a precursor to a cancerous state,although not in all patients, so their detection by means of the presentinvention may be used to provide valuable results for closer follow-up.In inflammatory bowel disease there may be sloughing of cells of thecolon and bowel, allowing for analysis to be performed on faecal samplesand preparations of cells from such samples. Example 32 below describesstaining of faecal smears prepared by recovering cells from faeces.

The present invention may be used to pre-screen samples before furtheranalysis. The present invention may be used for screening or analysis ofsamples previously tested using an available technique, such as a Papsmear test or ThinPrep 2000 test. The experiments below also show that aPap stain analysis and an analysis in accordance with the presentinvention, using an appropriate antibody, can be performed on the samepreparations. Thus, a cervical smear for example may be tested usingboth the conventional Pap smear test and a test in accordance with thepresent invention.

A further aspect of the present invention provides a method of markingabnormal cells within a tissue sample, the method including contactingthe sample with a specific binding member directed against a targetpolypeptide, such as Cdc6, MCM5 or other MCM as discussed, underconditions wherein the specific binding member binds to abnormallyproliferating cells and not normal cells. Whether or not the specificbinding member binds to the sample may be determined in order toascertain the presence of abnormally proliferating cells within thesample.

In a further aspect the present invention provides the use of a specificbinding member directed against a target polypeptide, as discussed, fordetermining, assessing or diagnosing the presence or absence of abnormalcellular proliferation, cellular growth abnormality, dysplasia,neoplasia, or a potentially or actually pre-cancerous or cancerous statein a tissue or sample thereof.

A specific binding molecule may be provided in a kit, which may includeinstructions for use in accordance with the present invention. Such kitsare provided as a further aspect of the present invention. One or moreother reagents may be included, such as labelling molecules, and so on(see below). Reagents may be provided within containers which protectthem from the external environment, such as a sealed vial. A kit mayinclude one or more articles for providing the test sample itselfdepending on the tissue of interest, e.g. a swab for removing cells fromthe buccal cavity, a syringe for removing a blood sample, a spatula fortaking a cervical smear, a biopsy gun and so on (such componentsgenerally being sterile). A kit may include any, any combination of orall of a blocking agent to decrease non-specific staining, a storagebuffer for preserving binding molecule activity during storage, stainingbuffer and/or washing buffer to be used during antibody staining, apositive control, a negative control and so on. Positive and negativecontrols may be used to validate the activity and correct usage ofreagents employed in accordance with the invention and which may beprovided in a kit. Controls may include samples, such as tissuesections, cells fixed on coverslips and so on, known to be eitherpositive or negative for the presence of the target, such as Cdc6 orMCM5. The design and use of controls is standard and well within theroutine capabilities of those of ordinary skill in the art.

Samples may be removed from the body using any convenient means andtechnique. For cervical screening, standard smear samples may beemployed. Alternatively, the ThinPrep 2000 technology (Cytec Corp,Boxborough, Mass., USA) may be used. This has been cleared by the US FDAas a replacement for the conventional method of Pap smear preparation. Asample is collected into a liquid medium instead of smearing the cellsonto a glass slide. An automated processor (the ThinPrep 2000 machine)is later used to collect cells from the liquid and deposit them in athin layer on a glass slide for analysis. A spatula or swab may be usedto remove endothelium cells, e.g. from the cervix or buccal cavity.Blood and other fluid samples may be removed using a syringe or needle.Other tissue samples may be removed by biopsy or tissue section.

In preferred embodiments the sample is not subject to antigen retrievalor pressure cooking/autoclaving. Antigen retrieval has long beenstandard in the art and is well known to those of ordinary skill.Hiraiwa et al. refer to Shin et al. (1991) Lab. Invest. 64, 693–702,which provides an exemplary approach. Samples may be fresh or frozen butare generally not formalin fixed or paraffin embedded. As discussed, ina particularly preferred embodiment the sample is a cervical smear.Cervical smears are not robust enough to be subject to pressure cooking.Furthermore, antigen retrieval treatment is generally not conducive toscreening where a high through-put is desirable.

Experimental exemplification of aspects of the present inventionincluded herein demonstrates applicability to the cervix, includingtesting cervical smears, the breast, urinary tract malignancies (testedon both biopsy tissue samples and on urine cytology smears), colon,lung, bladder, skin, larynx, oesophagus, bronchus, lymph nodes, andhaematological malignancies, also blood and serum for evidence ofmetastatic sarcoma and carcinoma. The present invention may additionallybe employed in assessment of pre-malignant abnormalities of cervicalglandular epithelial cells (glandular intra-epithelial neoplasia, GIN)or pre-malignant abnormalities in other tissues. It may be particularlyappropriate for employment in cytological or biochemical assessment ofother clinical specimens where detection of neoplastic cells, or theirdistinction from cells showing reactive changes, can be very difficult.Such specimens include sputum, bronchio-alveolar lavage specimens, urineand brushings from the alimentary tract (including oesophagus, stomachand pancreas, both bile duct and pancreatic duct). The present inventionmay be applied in histological or biological assessment of tissue whereassessment of proliferation may enable more accurate prediction ofclinical outcome, and/or more rational selection of therapy. Specimensmay include malignancies of glandular cells (eg. lung, breast, colon,prostate, stomach), squamous cells (eg. lung, skin, oesophagus) or otherepithelial cell types (eg. bladder, ureter, kidney, ovary).

The high degree of specificity observed in the experiments describedbelow with anti-Cdc6 antibodies and anti-MCM antibodies, includingvarious anti-MCM2, anti-MCM3, anti-MCM4, anti-MCM5, anti-MCM6 andanti-MCM7 antibodies, tested on a range of breast cancers provides forimmunocytological and biochemical approaches for diagnosis of breastcancer. Such may be applied to breast biopsies or fine needle aspiration(FNA) specimens or samplings of fluid from breast ducts, allowing foruse in screening programmes.

Samples to be subjected to a contact with a binding member in accordancewith various aspects of the present invention may be prepared using anyavailable technique which allows binding of a specific binding moleculeto the target polypeptide, such as Cdc6, MCM5 or other MCM as discussed,determination of nucleic acid levels, enzymatic activity and so on, inaccordance with different embodiments of the present invention. Varioustechniques are standard in the art, e.g. (for molecules such asantibodies binding target polypeptide) as used in fixing cells for thePap test.

The reactivities of a binding member such as an antibody on normal andtest samples may be determined by any appropriate means. Tagging withindividual reporter molecules is one possibility. The reporter moleculesmay directly or indirectly generate detectable, and preferablymeasurable, signals. The linkage of reporter molecules may be directlyor indirectly, covalently, e.g. via a peptide bond or non-covalently.Linkage via a peptide bond may be as a result of recombinant expressionof a gene fusion encoding binding molecule (e.g. antibody) and reportermolecule.

One favoured mode is by covalent linkage of each binding member with anindividual fluorochrome, phosphor or laser dye with spectrally isolatedabsorption or emission characteristics. Suitable fluorochromes includefluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed. Further examples are horseradish peroxidase andchemiluminescence.

The mode of determining binding is not a feature of the presentinvention and those skilled in the art are able to choose a suitablemode according to their preference and general knowledge.

In the experiments described below, horseradish peroxidase has beenemployed. Further experiments have been performed using alkalinephosphatase, with similar results being achieved (for instance withcervical smears). Alkaline phosphatase may be a more sensitive detectionsystem than horseradish peroxidase, but the developed colour is lesscompatible with PAP staining.

One protocol for antibody staining of cervical smears, which has beenemployed in embodiments of the present invention is as follows

-   1. Fix fresh smear for 5 minutes in 50:50 acetone:methanol. (Note    than an alternative starting point where a smear has been previously    fixed with “Cytofix”—an alcoholic and wax treatment that is in    standard use in the UK to treat smear samples when taken to allow    safe transport to a screening centre—is to remove Cytofix by soaking    in methylated spirits for 10 minutes. Should a smear have been    covered with any other protective layer, any appropriate treatment    may be employed to expose a sample to antibody staining.)-   2. Wash in Tris-buffered saline (TBS) for 5 minutes.-   3. Wash to permeabilise in 4 mM sodium deoxycholate in TBS for 15    minutes.-   4. Wash in TBS plus 0.3% Triton X100 for 5 minutes.-   5. Repeat step 4.-   6. Wash in TBS plus 0.025% Triton X100 for 5 minutes.-   7. Drain excess liquid without allowing tissue to dry out.-   8. Transfer slides into a humidified chamber and place on each slide    200 microlitres of 10% goat serum reagent in TBS for a minimum of 2    hours (or overnight).-   9. Drain excess liquid without allowing tissue to dry out.-   10. Place 200 microlitres of primary antibody diluted in TBS    containing 0.1% Triton and 1% BSA onto each slide and leave    overnight at 4° C. on an orbital shaker.-   11. Transfer slides into racks and wash in TBS plus 0.3% Triton X100    for 5 minutes.-   12. Wash in TBS plus 0.025% Triton X100 for 5 minutes.-   13. Repeat step 12.-   14. Drain excess liquid without allowing tissue to dry out.-   15. Transfer slides into a humidified chamber and place on each    slide 200 microlitres of biotinylated goat anti-rabbit secondary    antibody (Dako) at 1:500 in TBS containing 1% BSA for 1 hour.-   16. While slides are in secondary antibody, make up SABC solution.-   17. Transfer slides into racks and wash in TBS for 5 minutes.-   18. Place slides in endogenous peroxidase blocking agent wth 0.6%    hydrogen peroxide for 10 minutes.-   19. Wash in TBS for 5 minutes.-   20. Repeat step 19 twice.-   21. Transfer slides into a humidified chamber and place on each    slide 200 microlitres of SABC solution for 30 minutes.-   22. Transfer slides into racks and wash in TBS for 5 minutes.-   23. Repeat step 22.-   24. Develop in DAB solution for 10 minutes.-   25. Wash in running tap water for 5 minutes.-   26. Place slides in Harris' haematoxylin solution for 6 seconds.-   27. Wash in running tap water for 1 minute.-   28. Differentiate in 0.5% hydrochloric acid for 1–2 seconds.-   29. Wash in running tap water for 5 minutes.-   30. Rinse in 50% methanol for 2 minutes.-   31. Rinse in 70% methanol for 2 minutes.-   32. Rinse in 90% methanol for 2 minutes.-   33. Rinse in 100% methanol for 2 minutes.-   34. Place in Orange G working solution for 2 minutes.-   35. Rinse in 100% methanol for 7 seconds and agitate gently.-   36. Repeat step 35.-   37. Place in EA50 solution for 2 minutes.-   38. Rinse in 100% methanol for 7 seconds and agitate gently.-   39. Repeat step 38.-   40. Place slides in xylene to clear for 5 minutes.-   41. Repeat step 40 twice.-   42. Apply coverslips using DEPEX mountant.

Smears for immunofluorescence may be prepared in a similar fashion (andhave been). After the secondary antibody, they are incubated instrepavidin FITC-conjugated antibody for 1 hour and counterstained forDNA with propidium iodide/RNAse A (both Sigma at 50 ng/ml), then washedand mounted in glycerol/PBS/phenylenediamine.

Preferred binding molecules for use in aspects of the present inventioninclude antibodies, natural ligands for the target, small moleculeswhich target one or more epitopes on the target and T-cell Receptorbinding domains.

Antibodies which are specific for a target of interest, such as Cdc6,MCM5 or other MCM, may be obtained using techniques which are standardin the art. Methods of producing antibodies include immunising a mammal(e.g. mouse, rat, rabbit, horse, goat, sheep, monkey or bird such aschicken) with the protein or a fragment thereof or a cell or virus whichexpresses the protein or fragment. Immunisation with DNA encoding thetarget polypeptide is also possible. Antibodies may be obtained fromimmunised animals using any of a variety of techniques known in the art,and screened, preferably using binding of antibody to antigen ofinterest. For instance, Western blotting techniques orimmunoprecipitation may be used (Armitage et al, 1992, Nature 357:80–82).

The production of monoclonal antibodies is well established in the art.Monoclonal antibodies can be subjected to the techniques of recombinantDNA technology to produce other antibodies or chimeric molecules whichretain the specificity of the original antibody. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of an antibody to theconstant regions, or constant regions plus framework regions, of adifferent immunoglobulin. See, for instance, EP184187A, GB 2188638A orEP-A-0239400. A hybridoma producing a monoclonal antibody may be subjectto genetic mutation or other changes, which may or may not alter thebinding specificity of antibodies produced.

As an alternative or supplement to immunising a mammal with a peptide,an antibody specific for a target may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using lambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO92/01047. The library may be naive, that is constructedfrom sequences obtained from an organism which has not been immunisedwith the target or may be one constructed using sequences obtained froman organism which has been exposed to the antigen of interest (or afragment thereof).

Antibodies may be modified in a number of ways. Indeed, unless contextprecludes otherwise, the term “antibody” should be construed as coveringany specific binding substance having an antibody antigen bindingdomain. Thus, this covers antibody fragments, derivatives, andfunctional equivalents, including any polypeptide comprising animmunoglobulin binding domain, whether natural or synthetic. Chimaericmolecules comprising an immunoglobulin binding domain, or equivalent,fused to another polypeptide are therefore included. Cloning andexpression of chimaeric antibodies are described in EP-A-0120694 andEP-A-0125023.

It has been shown that the function of binding antigens can be performedby fragments of a whole antibody. Example binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544–546 (1989)) which consistsof a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al, Science, 242, 423–426, 1988; Huston etal, PNAS USA, 85, 5879–5883, 1988); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; P.Holliger et al Proc. Natl. Acad. Sci. USA 90 6444–6448, 1993).

Recombinant expression of polypeptides, including antibodies andantibody fragments, is well-known in the art.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells and many others. A common, preferred bacterial host is E. coli.The preferred hosts for baculovirus expression are insect cells such asthe SF9 cell line.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al, 1989, Cold Spring Harbor Laboratory Press. Transformationprocedures depend on the host used, but are well known.

Following production by expression from encoding nucleic acid anantibody or other specific binding molecule directed against a targetuseful in the present invention, such as Cdc6, may be recovered and maybe isolated, if necessary conjugated to an appropriate label orreporter, and provided for use in determination of the presence orabsence of cellular growth abnormality in a tissue sample, such as acervical smear, in accordance with the present invention as disclosed.

Levels of Cdc6 and MCM expression in tumours are much higher than innormal tissues and these antigens may be liberated into the bloodstream(e.g. due to necrosis of tumour cells) or other body fluids, e.g. urine,or faeces. A specific binding molecule may be used to detect the targetin a body fluid, e.g. serum, employing any technique available to thoseskilled in the art, such as DELFIA, ELISA, RIA, Western blotting. Tumourprogression and regression may be monitored, for instance in response totreatment or in relapse. Thus a blood or other bodily fluid sample, e.g.urine, prostatic fluid, nipple fluid, serous and ascitic effusions,cerebro spinal fluid, also faeces, may be assessed in accordance withthe present invention. For instance, a blood sample may be assayed forthe presence of a target polypeptide such as MCM5 and CDC6 using DELFIA,ELISA, RIA e.g. as described in Williams et al. Clin. Chem. Acta, 1986,155, 329–344.

Determination of binding to target in vivo may be used to identifylocalisation of abnormal cells in the body. Labelled binding moleculesagainst a target in accordance with the present invention may beadministered to an individual and binding within the body determined.When a radionucleotide such as Iodine-125, Indium-111, Thallium-201 orTechnetium-99m is attached to an antibody, if that antibody localisespreferentially in tumour rather than normal tissues, the presence ofradiolabel in tumour tissue can be detected and quantitated using agamma camera or scintigraphy. The quality of the tumour image obtainedis directly correlated to the signal:noise ratio. Radiolabelling withtechnetium-99m is described in Pak et al (1992), Nucl. Med. Biol. 19;699–677. A review of cancer imaging with anti-CEA antibodies is providedby Goldenberg D. M., Int. J. of Biol. Markers 1992, 7; 183–188. Shouldany method practised on the human or animal body be a method of actualdiagnosis of a disease, the present invention of course extends tospecific binding members directed against target polypeptides asdisclosed, for use in any such method.

ATPase enzymatic activity has been reported for Cdc6 and for MCMproteins. (Zwerschke et al., 1994, J. Biol. Chem. 269, 23351–23356;Ishimi et al. Cold Spring Harbor Meeting on Eukaryotic DNA Replication,Sep. 3–7 1997). These proteins may have other enzymatic activities, forinstance helicase activity as reported by Ishimi et al., (Ibid.). Thelevel of a target protein in accordance with the present invention maybe assessed by means of determination of its enzymatic activity in asample. For instance, specific chromogenic substrates have beendeveloped for the enzymatic activity of enzymes such as horseradishperoxidase (diaminobenzidine) and β-galactosidase (X-GAL).

Cdc6, MCM5 or other target protein expression may be assessed at thenucleic acid level, for instance by determining mRNA levels. Williams etal., (Cold Spring Harbor Meeting On Eukaryotic DNA Replication, Sep. 3–71997) have reported expression of Cdc6 mRNA at the base of theintestinal crypts of the mouse gut. Methods for RNA detection are wellknown in the field, and include Northern blotting, dot blotting, in situhybridisation, quantitative RT-PCR. Nucleic acid isolated and/orpurified from one or more cells or a nucleic acid library derived fromnucleic acid isolated and/or purified from cells (e.g. a cDNA libraryderived from mRNA isolated from the cells), may be probed underconditions for selective hybridisation and/or subjected to a specificnucleic acid amplification reaction such as the polymerase chainreaction (PCR). Binding of a probe to target nucleic acid may bemeasured using any of a variety of techniques at the disposal of thoseskilled in the art. For instance, probes may be radioactively,fluorescently or enzymatically labelled. Other methods not employinglabelling of probe include examination of restriction fragment lengthpolymorphisms, amplification using PCR, RNase cleavage and allelespecific oligonucleotide probing.

For practical purposes, or at least commercial purposes bearing in mindcost and time, assessment of target protein expression at the proteinlevel is generally preferred over assessment at the nucleic acid level.

Aspects of the present invention will now be illustrated with referenceto experimental exemplification. Further aspects and embodiments of thepresent invention will be apparent to those skilled in the art.

EXAMPLE 1 Preparation of Antibodies

Antibodies for Cdc6 were prepared using the following protocol.

Expressed sequence tags encoding fragments of human Cdc6-like protein(GenBank accession numbers: T90351, H59204, N69246, AA045217, AA099980)were identified on the basis of their weak sequence homology to humanOrc1 and yeast Cdc6/Cdc18. Corresponding library clones were obtainedfrom IMAGE Consortium (Research Genetics Inc., USA) and sequenced onboth strands using ABI PRISM 377 sequencer (Applied Biosystems). Aconsensus sequence of all five clones contained an open reading frame of536 amino acids. Alignment with the subsequently published sequence ofhuman Cdc6 (Williams et al., 1997) revealed 99.7% homology at theprotein level. Two separate fragments of human Cdc6 corresponding toamino acids 145–360 and 364–547 were cloned as Xbal-BamHI fragments intopET23a expression vector (Novagen) and expressed in E. coli CL41 strain.Recombinant protein fragments were purified by Ni+2 agarose affinitychromatography (Qiagen) and used for immunization. Antibodies wereraised and affinity purified as previously described (Romanowski et al.,Proc. Natl. Acad. Sci. USA, 1996, 93: 10189–10194). Three antibodiesrecognised a predominant band of 62 kD in HeLa total cell extracts andnuclear extracts.

Antibodies to MCM5 were prepared as follows:

PCR primers were designed on the basis of the published sequence ofhuman MCM5 (Hu et al., 1993, Nucl. Acid Res. 21 5289–5293). A fragmentof the MCM5 coding sequence corresponding to amino acids 367–582 wasamplified from reverse-transcribed HeLa cDNA and cloned as an SphI-BglIIfragment into pQE70 expression vector (Qiagen). The protein wasexpressed in BL21 E. coli cells and purified using Ni²⁺ agarose affinitychromatography (Qiagen). Antibodies were raised as described inRomanowski et al., Proc. Natl. Acad. Sci. USA, 1996, 93: 10189–10194.

EXAMPLE 2 Relationships of Cdc6 and MCM5 to Cell Proliferation

It has previously been shown by Western blot that the immortal humancell line HeLa expresses Cdc6 and MCM5 throughout the cell cycle(Williams et al., Proc. Natl. Acad. Sci. USA, 1997, 94: 142–147; Schulteet al., Eur. J. Biochem., 1996, 235, 144–151). Recently thedown-regulation of Cdc6 in quiescent Wi38 human fibroblasts was reportedby Williams et al, 1997.

The present inventors have also shown by Western blot that Cdc6expression is down-regulated when mouse 3T3 fibroblasts are madequiescent by contact inhibition.

The NIH 3T3 cell line was arrested by growing to confluence. Cultureswere held in quiescence for 7 days. Cells were released from G0 arrestby trypsin detachment and replating. Soluble (supernatant) and nuclearprotein (pellet) extracts were prepared at three-hourly intervals andboth extracts were immunoblotted with antibodies against human MCM5,Orc2 and Cdc6. Orc2 (see Gavin et al. for cloning—Science (1995) 2701667–1671) remains chromatin bound in quiescent cells (G0) and does notincrease appreciably as cells enter the cell cycle. In contrast MCM5could not be detected in the chromatin bound fraction (pellet) ofquiescent cells even though the soluble fraction contained significantamounts of MCM5. In contrast to MCM5, Cdc6 was completely absent fromquiescent cells but the expression of this protein was rapidly inducedas cells re-entered the cell cycle. Similar results were also obtainedwith the human EJ13 cell line (derived from a bladder carcinoma).

These results provide indication that the absence of Cdc6 in quiescentcells is a general phenomenon.

These studies were extended by immunofluorescence and application ofanti-Cdc6 and anti-MCM5 antibodies to whole cells.

Expression of Cdc6 and of MCM5 was found to be also down-regulated whennewborn human fibroblasts (NHF) are made quiescent by contactinhibition. NHF were grown to confluence and held in quiescence forthree days. Cells were released from G0 arrest by trypsin detachment andreplating. Whole cells were then harvested at multiple time points afterrelease up until entry into S-phase. Staining with propidium iodide wasused to reveal DNA and results were compared with staining of thesamples with anti-Cdc6 and anti-MCM5 antibodies.

Quiescent (G0) cells showed no Cdc6 immunoreactivity, and a very weaksignal with anti-MCM5 antibody. However, during entry into cell cycleand S-phase, strong nuclear immunoreactivity for Cdc6 and MCM5 wasobserved.

These studies might suggest that anti-Cdc6 antibodies could provide amarker for proliferating cells similar to PCNA or Ki67. Neither PCNA norKi67 has proved satisfactory for cervical cytology.

EXAMPLE 3 Anti-Cdc6 and Anti-MCM5 Binding Molecules Detect Abnormal(e.g. Tumour) Cells Much More Effectively than PCNA or Ki67 Antibodies

Results outlined in Example 2 suggest that Cdc6 expression mightresemble that of PCNA or Ki67, neither of which has proved satisfactoryfor cervical cytology. The present inventors compared antibodies tothese proteins on sections of normal or diseased uterine cervix.

Immunostaining of cervical SILs for the conventional proliferationmarkers PCNA and Ki67 shows different pattern of immunoreactivity whencompared with staining for Cdc6 or MCM5. In normal cervix, antibodiesagainst all four antigens showed positive immunostaining of epithelialcells confined to the basal and parabasal layers. No immunostaining ofmetaplastic, stromal or inflammatory cells was observed. In both low andhigh grade SILs (LSIL and HSIL) antibodies against Cdc6 and Mcm5 showedpositive immunostaining of the majority (>95%) of the abnormal cells. Incontrast, immunostaining for PCNA and Ki67 was positive in only aminority population (<30%) of abnormal cells in both grades of SILs.Koilocytes, which are characteristic of LSIL and reflect an HPVcytopathic effect, all showed positive immunostaining with Cdc6 andMCM5, whereas only a minority population (20%) showed positive stainingfor PCNA or Ki67.

The much greater level of staining of abnormal cells provides a distinctadvantage for using anti-Cdc6 or anti-MCM5 binding molecules overanti-PCNA and anti-Ki67 molecules in cervical screening wherein tissuesamples are most readily taken by way of smears from the surface of theepithelium.

5 μm sections were cut onto glass slides and de-waxed in xylene.Endogenous peroxidase activity was quenched by incubation in 0.6%hydrogen peroxide in 100% methanol for 3–5 minutes at room temperature.The slides were washed in phosphate-buffered saline for 2×5 minutes andthen blocked with approximately 100 μl per section of 10% foetal calfserum (FCS) in phosphate buffered saline (PBS). The slides were drainedand excess serum wiped away. Primary antibodies were diluted to 1 in 20in PBS containing 2.5% FCS, and 25–5 μl was added to each section.Incubation was for 45 minutes at room temperature in a humidifiedchamber. The slides were then washed for 3×5 minutes in PBS, followed byblocking with 20% rabbit serum for anti-Ki67 or anti-PCNA and 20% donkeyserum for anti-Cdc6 or anti-MCM5 in PBS for 15 minutes. After drainingthe blocking antibody and wiping the slides, biotinylated rabbitanti-mouse secondary antibody (for anti-Ki67 or anti-PCNA) or donkeyanti-rabbit (for anti-Cdc6 or anti-MCM5) at 1 in 200 in PBS containing10% normal human serum was added for 30 minutes at room temperature.After 3×5 minute washes in PBS, streptavidin-biotin-horseradishperoxidase complex was added at 1 in 500 in PBS for 30 minutes at roomtemperature. Following 3×5 minute PBS washes the substratediaminobenzidine was added at 1% in 100 mM Tris.Cl (pH 7.6) containing0.005% hydrogen peroxide, and incubated at room temperature for 5minutes. The reaction was stopped by rinsing in tap water, and slideswere lightly stained with Gill's haematoxylin, dehydrated through gradedethanols and washed 2×6 minutes in xylene. Coverslips were applied withDPX mounting medium.

Serial sections of normal cervix were stained for PCNA, Ki67, MCM5 andCdc6 using respective antibodies. All these antibodies showed similarpatterns of immunoreactivity with positive cells confined to the basaland parabasal layers only.

Serial sections of a low grade SIL (CINI) cervix were stained for PCNA,Ki67, MCM5 and Cdc6 respectively using the antibodies. The dysplasia isprominent in the lower third of the squamous epithelium and isassociated with HPV related viral changes (koilocytosis), the latterextending to the surface. PCNA and Ki67 show patchy focalimmunoreactivity which is confined to the lower ⅓ of the epithelium andin which only a small proportion of the atypical cells are positivelystained. In contrast Cdc6 and MCM5 show full thickness immunoreactivitywith positive staining of all atypical cells including the koilocytes inthe more superficial layers of the epithelium.

Serial sections of a high grade SIL were stained using PCNA, Ki67, MCM5and Cdc6 antibodies respectively. Dysplasia is present throughout alllayers of the epithelium. PCNA and Ki67 show similar patterns ofimmunoreactivity with full thickness staining but in which only aminority population of the atypical cells are positive (up toapproximately 30%). Cdc6 and MCM5 also show full thickness staining ofthe epithelium. However, in marked contrast to PCNA and Ki67, Cdc6 andMCM5 show positive staining of all atypical cells.

These results are indicative of the particular usefulness of usinganti-Cdc6 or anti-MCM5 specific binding molecules in assessing the stateof a cervix by means of determining binding to a smear sample. Smearssample only a top surface layer of cells from the cervix, so it isimportant to have a high level of staining. Such high-level stainingobtained with anti-Cdc6 and with anti-MCM5 on the early stageabnormality (low grade SIL, CINI), not shown with anti-PCNA noranti-Ki67, is very significant. Furthermore, the full thickness stainingobtained using anti-Cdc6 and anti-MCM5 antibodies on LSIL samples, butnot anti-PCNA nor anti-Ki67 antibodies, highlights the particularusefulness of the former for assessing smear samples for early stagepotential pre-malignancy.

EXAMPLE 4 Cdc6 and MCM5 Antibodies Detect Abnormal Cells In CervicalSmears

Example 3 demonstrates the value of anti-Cdc6 and anti-MCM5 bindingmolecules for detecting potentially pre-malignant lesions in sections ofthe uterine cervix. Further experimental results show that they areequally effective in detecting abnormal cells in cervical smearpreparations.

Cervical smears were fixed for 10 minutes in formaldehyde (4% freshlyprepared from paraformaldehyde in phosphate-buffered saline). The fixedmaterial was then stained with anti-Cdc6 antibodies (1:200) or anti-MCM5antibodies (1:200) followed by donkey anti-rabbit polyclonal antibodyconjugated to fluorescein isothiocyanate (Amersham; 1:100). Total DNAwas labelled by propidium iodide. Images were obtained using scanninglaser confocal microscopy (Bio-Rad MRC 1024). In these images total DNAis red, Cdc6 or MCM5 immunostaining is green and so immunoreactivenuclei appear yellow.

Examples of normal cervical smears showed a characteristic strip ofparallel arranged endocervical cells and a mixed population ofsuperficial and metaplastic squamous cells. There was no evidence ofspecific anti-Cdc6 or anti-MCM5 immunoreactivity with any of theantibodies tested.

Abnormal smears containing dyskaryotic cells (atypical squamous cells)showed positive staining with three different anti-Cdc6 antibodies andwith an anti-MCM5 antibody. Koilocytes also showed strongimmunoreactivity with anti-Cdc6 and anti-MCM5 antibodies. None of theadjacent normal superficial squamous/metaplastic cells shows Cdc6 orMCM5 immunoreactivity.

Results were obtained using different anti-Cdc6 antibodies,preferentially staining LSIL cells, including koilocytes, against a verylow background in smears of normal cervix, or in normal cells in smearsfrom abnormal cervix. These results were also obtained using ananti-MCM5 antibody.

Similar results were seen with antibodies against MCM5.

EXAMPLE 5 Cdc6 and MCM5 Antibodies Preferentially Stain Cancer Cells inthe Breast

Anti-Cdc6 and anti-MCM5 antibodies were tested on another site of commoncancers, the breast. Breast tissue fixing and staining was as describedin Examples 3 and 4 for cervical smears.

Anti-Cdc6 and anti-MCM5 antibodies were tested on a range of breastcancers.

Whereas the normal breast showed no evidence of immunostaining, strongpositive staining was observed with both anti-Cdc6 and anti-MCM5antibodies in a variety of histological types of breast cancer includinglow and high grade invasive ductal carcinoma. A low grade mucosalcarcinoma also showed strong positive staining with both antibodies.Importantly, normal stromal cells adjacent to cancer were negative.

EXAMPLE 6 Analysis of Blood Samples

Archival blood samples from patients with disseminated metaplasticdisease are assayed for the presence of MCM5 and CDC6 using anenzyme-linked immunosorbent assay as described in Williams et al. Clin.Chem. Acta, 1986, 155, 329–344. The amount of soluble MCM5 and CDC6 inserum is correlated with tumour load.

EXAMPLE 7 Comparison of Smears and Frozen Sections with Paraffin WaxEmbedded Tissue Sections with Antigen Retrieval

Immunoperoxidase staining of 5 μm sections of formalin fixed paraffinwax embedded tissue sections of normal cervix (seven samples), LSIL(five samples), HSIL (six samples) and squamous cell carcinoma (sixsamples) was performed with antibodies against Ki67, PCNA, MCM5 andCdc6.

5 μm sections were cut onto glass slides and de-waxed in xylene.Endogenous peroxidase activity was quenched by incubation in 0.6%hydrogen peroxide in 100% methanol for 30 minutes at room temperature.The slides were washed for 2 minutes in ultrapure water and thenpressure cooked for 2 minutes in sodium citrate buffer. The slides werewashed in Tris buffered saline (TBS) for 2×5 minutes and then blockedwith approximately 100 μl per section of 10% goat serum in TBS. Theslides were drained and excess serum wiped away. Primary antibodies werediluted to 1 in 200 in TBS containing 1% BSA, and 100 μl was added toeach section. Incubation was overnight at 4° C. in a humidified chamber.The slides were then washed for 3×5 minutes in TBS, followed bybiotinylated goat anti-rabbit secondary antibody at 1 in 500 in TBScontaining 1% BSA for 30 minutes at room temperature. After 3×5 minutewashes in TBS, streptavidin-biotin-horseradish peroxidase complex wasadded at 1 in 500 in TBS for 30 minutes at room temperature. Following3×5 minute TBS washes the substrate diaminobenzidine was added at 1% inTBS containing 0.005% hydrogen peroxide, and incubated at roomtemperature for 10 minutes. The reaction was stopped by rinsing in tapwater, and slides were lightly stained with haematoxylin, dehydratedthrough graded ethanols and cleared in xylene. Coverslips were appliedwith DPX mounting medium.

Immunoperoxidase staining of frozen tissue sections of normal cervix(eight samples), HSIL (nine samples) and LSIL (eight samples) wasperformed with antibodies against PCNA, Ki67, MCM5 and Cdc6.

Frozen sections were fixed for 10 minutes in acetone. Endogenousperoxidase activity was quenched by incubation in 0.6% hydrogen peroxidein 100% methanol for 30 minutes. Sections were then washed in TBS andblocked overnight with 10% goat serum in TBS. Primary antibodies werediluted 1/200 in TBS containing 1% BSA and incubated overnight at 4° C.The secondary antibody procedure was then followed as described abovefor fixed tissue sections.

The sensitivity of staining with anti-MCM5 and anti-Cdc6 antibodies wasmuch higher than with anti-PONA and anti-Ki67 antibodies when applied tothe frozen sections.

On the normal, LSIL and squamous cell carcinoma tissue fixed informalin, paraffin embedded and exposed to pressure cooking, anti-PCNA,anti-MCM7, anti-MCM5 and anti-Cdc6 gave similar patterns of staining.Ki67 in comparison was much less sensitive with only focal weak stainingof LSIL and squamous cell carcinoma.

EXAMPLE 8 Staining with Anti-MCM7 Antibodies On Frozen Sections and OnTissues Subject To Antigen Retrieval

Four frozen sections of cervix classified as HSIL were stained withanti-MCM7 antibody.

Staining was observed with the same pattern as for staining withanti-Cdc6 and anti-MCM5 antibodies.

This result differs from that of Hiraiwa et al. (Int. J. Cancer, 1997,74: 180–184) who found similar immuno-staining patterns for PCNA andMCM7 (hCDC47) in several human tissues and three types of human tumourwhich had been subjected to antigen retrieval protocols.

However, consistent with Hiraiwa et al., staining patterns obtained foranti-MCM7 antibodies on normal cervix, LSIL and squamous cell carcinomaparaffin wax embedded tissue sections subject to antigen retrieval werefound to be similar to those obtained for anti-PCNA antibodies. Asindicated in Example 7, staining patterns for anti-MCM5 and anti-Cdc6antibodies on such sections so prepared were also similar to those foranti-PCNA antibodies.

EXAMPLE 9 Staining with Anti-MCM2 Antibodies

Rabbit polyclonal anti-human MCM2 was used to stain two frozen sectionsof normal cervix and four frozen sections of HSIL (each of whichincluded normal cervical epithelium).

Normal ectocervix showed staining of nuclei in the basal layer only,with no expression in superficial differentiated cells. In contrast,there was nuclear staining of HSIL cells throughout the full thicknessof the abnormal epithelium. Endocervical cells were negative.

EXAMPLE 10 Staining with Anti-MCM3 Antibodies

Rabbit polyclonal anti-human MCM3 was used to stain a frozen section ofnormal cervix and two frozen sections of HSIL, (each of which includednormal cervical epithelium).

Normal ectocervix showed rather granular staining of nuclei in the basallayer only, with no nuclear expression in superficial differentiatedcells. Some background staining of keratinocyte cytoplasm was seen. Incontrast, there was nuclear staining of HSIL cells throughout the fullthickness of the abnormal epithelium. There was some staining ofendocervical mucus, although the nuclei of endocervical cells werenegative.

Polyclonal anti-human MCM3 was also used to stain four smears of HSILand two smears of LSIL (each of which included normal cervical cells).There was nuclear staining of SIL cells in each case. In addition, therewas background cytoplasmic staining of keratinocytes and some stainingof endocervical cell nuclei at the dilution of primary antibody used.

Polyclonal anti-Xenopus MCM3 was used to stain a frozen section of HSIL.Cross-reactivity of the anti-Xenopus MCM3 antibodies with human MCM3 wasconfirmed by Western blotting and localisation on tissue sections. Therewas nuclear staining of HSIL cells throughout the full thickness of theabnormal epithelium.

EXAMPLE 11 Staining with Anti-MCM4 Antibodies

Rabbit polyclonal anti-human MCM4 was used to stain a frozen section ofnormal cervix and two frozen sections of HSIL (each of which includednormal cervical epithellum).

Normal ectocervix showed rather granular staining of nuclei in the basallayer only, with no nuclear expression in superficial differentiatedcells. Some background staining of keratinocyte cytoplasm was seen. Incontrast, there was nuclear staining of HSIL cells throughout the fullthickness of the abnormal epithelium, with strong staining of surfacenuclei. There was weak staining of endocervical cell nuclei at thedilution of primary antibody used.

Polyclonal anti-human MCM4 was also used to stain two smears of HSIL(each of which included normal cervical cells). There was nuclearstaining of HSIL cells in each case. In addition, there was backgroundcytoplasmic staining of keratinocytes and some staining of endocervicalcell nuclei at the dilution of primary antibody used.

EXAMPLE 12 Staining with Anti-MCM6 Antibodies

Rabbit polyclonal anti-human MCM6 was used to stain a frozen section ofnormal cervix and two frozen sections of HSIL (each of which includednormal cervical epithelium).

Normal ectocervix showed strong staining of nuclei in the basal layeronly, with no nuclear expression in superficial differentiated cells. Incontrast, there was strong nuclear staining of HSIL cells throughout thefull thickness of the abnormal epithelium. There was some staining ofendocervical mucus, but minimal staining of endocervical cell nuclei.

Polyclonal anti-human MCM6 was also used to stain four smears of HSILand four smears of LSIL (each of which included normal cervical cells).There was nuclear staining of SIL cells in each case. In addition, therewas background cytoplasmic staining of keratinocytes and some stainingof endocervical nuclei at the dilution of primary antibody used.

EXAMPLE 13 Further Staining Experiments with Anti-MCM7 Antibodies

Rabbit polyclonal anti-human MCM7 was used to stain three frozensections of normal cervix, six frozen sections of HSIL and a frozensection of a cervical SCC (each of which included normal cervicalepithelium).

Normal ectocervix showed staining of nuclei in the basal layer only,with no nuclear expression in superficial differentiated cells. Somebackground staining of keratinoctye ectoplasm was seen. In contrast,there was strong nuclear staining of the large majority of HSIL cellsthroughout the full thickness of the abnormal epithelium. There was somestaining of endocervical mucus, and weak staining of endocervical cellnuclei at the dilution of primary antibody used.

Polyclonal anti-human MCM7 was also used to stain two smears of HSIL andtwo smears of LSIL (each of which included normal cervical cells). Therewas nuclear staining of SIL cells in each case. In addition, there wasbackground cytoplasmic staining of keratinocytes and some staining ofendocervical cell nuclei at the dilution of primary antibody used.

Polyclonal anti-Xenopus MCM7 (confirmed to cross-react with human MCM7by Western blotting and localisation on tissue sections) was used tostain a frozen section of HSIL. There was nuclear staining of HSIL cellsthroughout the full thickness of the abnormal epithelium.

Methods

Preparation of Cervical Smears

Fresh smears were fixed (5 minutes in 50:50 acetone:methanol) and airdried. After quenching endogenous peroxidase activity (as above), cellswere permeabilised (4mM sodium deoxycholate for 10 minutes), washed (TBSwith 0.25% Triton X-100) and blocked overnight with 10% goat serum inTBS. Primary antibodies were diluted 1/200 in TBS containing 1% BSA andincubated overnight at 4° C. The slides were then washed 3×5 minutes inTBS, followed by biotinylated rabbit secondary antibody (Dako) at 1 in500 in TBS containing 1% BSA for 30 minutes at room temperature. After3×5 minute washes in TBS, streptavidin horseradish peroxidase complex(Dako) was added at 1 in 500 in TBS for 30 minutes at room temperature.Following 3×5 minute TBS washes the substrate diaminobenzidine was addedat 1% in TBS containing 0.005% hydrogen peroxide, and incubated at roomtemperature for 10 minutes. The reaction was stopped by rinsing in tapwater, and slides were lightly counter-stained with haematoxylin,dehydrated through graded ethanols and cleared in xylene. Coverslipswere applied with DPX mounting medium.

Immunofluorescence

Freshly collected cervical smear material was suspended in 0.5 ml PBSand fixed by addition of 0.5 ml 8% formaldehyde and spun onto polylysinecoverslips. Coverslips were processed as described in Romanowski et al.,Proc. Natl. Acad. Sci. USA, 1996, 93: 10189–10194. Following blocking in5% BSA/PBS/TritonX-100 and SDS they were incubated with primaryantibody, washed, incubated with secondary antibody (FITC conjugatedanti-rabbit antibody Amersham 1:100) and counterstained for DNA withpropidium iodide/RNAse A (both Sigma at 50 ng/ml), washed and mounted inglycerol/PBS/phenylenediamine.

Fluorescent images were collected on a BioRad MRC 1024 scanning laserconfocal microscope using a two channel (FITC & Texas Red) method. Forsome images confocal series at 1–2 μm steps were collected and thenprojected as a single frame (FIG. 4a and c). Normal and tumour breasttissue was collected freshly from mastectomy specimens. Thin slices (<1mm) were fixed in 4% paraformaldehyde for 30 minutes and then processedas above but allowing longer times for both antibody incubations andwashings.

EXAMPLE 14 A blinded Comparison of An Embodiment of the PresentInvention with Standard Pap Staining

A blinded trial was performed to compare detection efficiency usingantibodies against MCM5 with standard Pap staining performed on smearsobtained from females attending colposcopy outpatient clinics at localhospitals.

Table 1 shows that of 26 cases assessed as positive by routine Papstain, all 26 were also scored positive by the antibody test inaccordance with the present invention. Of 16 cases scored negative byroutine Pap stain, 13 were also scored negative by the antibody test.

Of the remaining three, one contained stained immature metaplasticsquamous cells showing reactive changes in an inflammatory background.The other two were confirmed as containing abnormal (LSIL) cells onre-examination of the Pap stain, i.e. were false-negatives of the kindwhich the present invention may be used to eliminate.

These results demonstrate no loss of information from the Pap stain,only a gain of information using an antibody test in accordance with thepresent invention. This allows for any possible failure of the antibodytest to be underwritten using the conventional Pap stain.

EXAMPLE 15 Analysis of Urine Samples of Patients with Urinary TractMalignancies

A Dissociation Enhancement Lanthanide Fluorescence Immunoassay(“DELFIA”) was established for detection of human MCM5 using twodifferent rabbit polyclonal anti-hMCM5 antisera.

The basis of the sandwich assay is the immobilisation of an excess ofspecific antibody to a surface (here polystyrene microtiter wells)—i.e.the “capture” antibody. Following the primary antibody-binding reactiona second (here europium) labelled antibody with a different epitopespecificity is added in excess. After the immunoreaction has beencompleted the excess materials are washed away and, following theaddition of enhancement solution, (Wallac Oy) time resolved fluorescenceis measured in a time resolved fluorometer. The signal is proportionalto the concentration of the analyte.

The following assay has been employed:

-   1. Coating with polyclonal rabbit anti MCM5 Ab (1600 ng/well)    overnight (4° C.);-   2. 3× Washing with DELFIA wash buffer (Wallac Oy);-   3. Blocking in 5% BSA/PBS for 1 hr;-   4. 3× Washing with DELFIA wash buffer (Wallac Oy);-   5. Primary antibody-binding reaction (1:3 dilution of analyte in    Wallac multi buffer with 0.02% TWEEN) overnight (4° C.);-   6. 4× Washing with DELFIA wash buffer (Wallac Oy);-   7. Secondary antibody-binding reaction with europium labelled    polyclonal rabbit anti-MCM5 Ab (4²⁰Eu/IgG) for 3 hrs;-   8. 6× Washing with DELFIA wash buffer (Wallac Oy);-   9. Addition of enhancement solution 10 min incubation with shaking.    Measurement of time resolved fluorescence in a time resolved    fluorometer (Wallac Oy).

Using polyclonal rabbit anti-MCM5 antisera from two different rabbitsand recombinant human MCM5 in 5% BSA/PBS as the analyte a standard curvebetween 13 pM and 41250 pM was achieved. The concentration of hMCM5 in aspecimen was determined by comparison of the DELFIA assay counts of thesample to a standard curve made from recombinant hMCM5 5% BSA/PBS. Evenbetter sensitivity is to be expected for when monoclonal antibodies areused.

Urine specimens from patients with urinary tract malignancies atAddenbrookes Hospital, Cambridge, UK, were centrifuged (50–150 ml) at3000 rpm (SIGMA 4K10, 7 min, 4° C.) and soluble fractions from the cellpellet were produced by hypotonically swelling, douncing and saltextraction of DNA-bound proteins. Soluble fractions were assayed usingthe MCM5 DELFIA and the biochemical data compared with diagnosticreports for urology pathology services at the hospital.

Of 5 samples determined clinically to be positive for malignancy, 4scored positive using the DELFIA in accordance with the invention (80%)showing measurable amounts of MCM5 (29–85 pM). Of 6 samples determinedclinically to be negative for malignancy, all gave responses similar tothe zero standard with the DELFIA.

EXAMPLE 16 Analysis of Blood Samples of Patients with Acute and ChronicLeukaemia/Lymphoma

The DELFIA described in Example 15 was used in testing blood samplesobtained from patients with acute and chronic leukaemia/lymphoma atAddenbrookes Hospital, Cambridge. Blood was centrifuged at 3000 rpm(SIGMA 4K10, 7 min, 4° C.) and soluble fractions from the cell pelletwere produced by hypotonically swelling, douncing and salt extraction ofDNA bound proteins. Soluble fractions were subsequently assayed usingthe DELFIA.

Of 6 malignant cases, 5 tested positive using the DELFIA in accordancewith the present invention (83%) showing measurable amounts of MCM5(24–1945 pM). Of 6 control samples (Blood from diabetic outpatients) allgave responses similar to the zero standard.

EXAMPLE 17 Serological Detection of Metastatic Malignancy

Assays were performed with serum from patients with metastatic breastand ovarian cancer at Addenbrookes Hospital, Cambridge, UK using theDELFIA described in Example 15.

Two Sarcoma cases and three carcinoma cases (breast and ovaryadenocarcinoma) showed measurable amounts of MCM5.

EXAMPLE 18 Preparation of “pan-MCM” Polyclonal Antibody for Use in theInvention

A polyclonal antibody preparation able to bind MCM2, MCM3, MCM4, MCM5,MCM6 and MCM7 was obtained as follows.

The peptide VVCIDEFDKMSDMRTAC (SEQ ID NO:1), corresponding to aconsensus sequence common to the MCM family of proteins, was synthesizedusing t-BOC chemistry. The peptide was conjugated to PPD (purifiedprotein derivative—tuberculin)

Rabbits were immunized by injection at 21 day intervals. 10 days afterthe third immunization serum was harvested and was used in subsequentexperiments (referred to as “pan-MCM” antibody in the followingexperimental examples).

EXAMPLE 19 Staining of Normal Breast and Breast Carcinoma Tissues withAnti-CDC6, Anti-MCM2, Anti-MCM5, Anti-MCM7 and pan-MCM Antibodies

Histological specimens of normal breast (recipients of breast reductionoperations) and biopsy-proven ductal and lobular carcinomas were stainedwith antibodies against CDC6, MCM2, MCM5 and MCM7 and a pan-MCMantibody. The anti-MCM2 antibody was the BM28 mouse monoclonal antibodyavailable commercially from Transduction Laboratories (see their 1998Antibody Catalog). Staining was performed individually for each antibodyas described.

Both formalin-fixed, paraffin embedded specimens which were the subjectof pressure cooking, and frozen specimens were examined.

Formalin fixed, paraffin embedded human tissues obtained for diagnosticbiopsy or after resection at Addenbrooke's Hospital were utilised inaccordance with ethical guidelines approved by the hospital. Five micronsections were cut from these tissues onto APES(aminopropyltriethoxysilane) coated slides, dewaxed in xylene and takenthrough alcohols to water. The tissue was pressure cooked in citratebuffer to facilitate epitope retrieval, following by washing inTris-buffered saline (TBS). Endogenous peroxidase activity was quenchedby incubation in 0.6% hydrogen peroxide in TBS for 30 minutes.

Sections were washed in TBS and blocked with 10% goat serum in TBS forup to 2 hours. Primary antibodies were diluted in TBS containing 0.1%Triton and 1% Bovine serum albumen (BSA). One hundred microlitres wasadded to each section and the slides were incubated at 4° C. in ahumidified chamber.

The slides were then washed in TBS containing 0.025% Triton, followed byincubation in biotinylated goat anti-rabbit secondary antibody (DAKO) at1:500 in TBS containing 1% BSA for 1 hour at room temperature. Afterwashes in TBS, a strepavidin horseradish peroxide system using thesubstrate diaminobenzidine was used to stain the slides. The reactionwas stopped by rinsing in water and lightly counter-stained with Harris'haematoxylin, dehydrated through graded ethanols and cleared in xylene.Coverslips were applied with DEPEX mounting medium.

Frozen sections were prepared as described above in Example 7 exceptblocking with 10% goat serum was for 30 minutes and not overnight.

In the normal breast tissue, only 1–3% of ductal and lobular cellsstained positive. Stromal cells were negative.

50–80% of abnormal cells in a variety of breast carcinomas, includinglow and high grade lesions and lobular and ductal type, showed positivestaining, with surrounding stromal cells and inflammatory cellsremaining unstained.

These results were obtained individually with each of the antibodies.

A comparison was made with anti-PCNA and anti-Ki67 antibodies. On theparaffin sections, anti-PCNA staining gave similar results to anti-MCM5and anti-CDC6 while anti-Ki67 antibodies gave only weak, focal staining.On the frozen sections, staining with anti-MCM and anti-CDC6 antibodiesgave much better results than either anti-PCNA or anti-Ki67 antibodies.

EXAMPLE 20 Staining of Normal Prostate and Adenocarcinoma of theProstate Using Antibodies Against MCM5, MCM7 and pan-MCM

Paraffin-embedded histological specimens of normal tissue andadenocarcinoma of the prostate, prepared as described for breast tissuein Example 19, were stained with anti-MCM5, anti-MCM7 and pan-MCMantibodies in separate experiments.

Normal cases showed positive staining of less than 10% of cells witheach antibody, whereas adenocarcinomas showed staining of 30–50% oftumour cells, with surrounding stromal cells and inflammatory cellsremaining unstained.

EXAMPLE 21 Staining of Normal Colon and Carcinoma of the Colon UsingAntibodies Against MCM2, MCM5, MCM7, pan-MCM and CDC6

Histological resection specimens of colon adenocarcinoma andtubulovillous adenoma were stained separately with antibodies againstMCM2, MCM5, MCM7, pan-MCM and CDC6. Normal specimens were also stainedwith these antibodies.

In normal tissue, the staining for each antibody was only seen in thelower third of colonic crypts, with more superficial differentiatedcells in the crypts remaining unstained.

In both tubulovillous adenoma and adenocarcinoma tissues, more than 50%of tumour cells were positive for staining with each antibody, with nostaining of surrounding connective tissue elements.

Both frozen and paraffin-embedded samples were examined, as for breasttissue in Example 19. The results were similar as between anti-MCM andanti-CDC6 antibodies on the one hand and anti-PCNA and anti-Ki67antibodies on the other, i.e. on frozen samples the staining withanti-MCM and anti-CDC6 antibodies was superior to that obtained usinganti-PCNA and anti-Ki67 antibodies.

EXAMPLE 22 Staining of Normal Tissue and Carcinoma of the Lung, withAntibodies Against MCM2, MCM5, MCM7 and pan-MCM

Paraffin embedded histology specimens of biopsies or resections frompatients with squamous cell carcinoma or adenocarcinoma of the lung werestained separately with anti-MCM2, anti-MCM5, anti-MCM7 and pan-MCMantibodies. The specimens were prepared as described for breast tissuein Example 19. Staining was compared with staining on normal parenchymallung tissue.

In the normal tissue, the stained proliferative fraction was very low.

In all carcinomas, more than 30% of tumour cells were positive, with nostaining of surrounding inflammatory or connective tissue cells.

EXAMPLE 23 Staining of Bladder, Both Normal and Carcinoma, withAnti-MCM2, Anti-MCM5, Anti-MCM7, pan-MCM and Anti-CDC6 Antibodies

Histological specimens from biopsies of transitional cell carcinomastaken at cystoscopy were stained with anti-MCM2, anti-MCM5, anti-MCM7,pan-MCM and anti-CDC6 antibodies.

In normal bladder tissue, there was strong staining of the basal layerof transitional epithelium with the more superficial differentiatedcells remaining unstained.

In fragments containing carcinoma in situ, the full thickness ofdysplastic cells stained positive.

Cases of invasive transitional cell carcinoma showed 50–100% nuclearstaining of tumour cells, with negative stromal and inflammatorycomponents.

Both frozen and paraffin-embedded samples were examined, as for breasttissue in Example 19. The results were similar as between anti-MCM andanti-CDC6 antibodies on the one hand and anti-PCNA and anti-Ki67antibodies on the other, i.e. on frozen samples the staining withanti-MCM and anti-CDC6 antibodies was superior to that obtained usinganti-PCNA and anti-Ki67 antibodies.

EXAMPLE 24 Staining of Various Skin Samples with Anti-MCM5 Antibodies

Histological samples from normal skin, hyperplastic conditions(including psoriasis), solar keratoses, Bowen's disease and invasivesquamous cell carcinomas were stained with anti-MCM5 antibodies.

Normal skin showed staining of predominantly the basal layer of theepithelium with occasional cells in the lower third of the epidermisalso staining, but more superficial differentiated cells remainingunstained.

In cases of psoriasis, there was more predominant staining in thelowermost 3–4 layers on the epidermis, reflecting the increased turnoverrate of the skin.

Solar keratoses and Bowen's disease (carcinoma in situ) showed stainingof all dysplastic cells in the epidermis, up to full thickness.

Invasive squamous cell carcinomas showed staining of greater than 70% ofcells, with well differentiated tumours showing small foci of negativedifferentiated cells adjacent to keratin pearls.

EXAMPLE 25 Staining of Larynx with Anti-MCM5 Antibodies

Histological samples of normal and carcinoma larynx, prepared as for theparaffin embedded specimens of breast tissue described in Example 19,were stained with anti-MCM5 antibody.

Normal cases showed staining of basal proliferating epithelial cellsonly (less than 10%).

Carcinomas showed greater than 50% cells with nuclear staining. Stromaland inflammatory cells were negative throughout.

EXAMPLE 26 Staining of Oesophagus with Anti-MCM5 Antibodies

Histological samples of normal and carcinoma oesophagus, prepared as forthe paraffin embedded specimens of breast tissue described in Example19, were stained with anti-MCM5 antibody.

Normal cases showed staining of basal proliferating epithelial cellsonly (less than 10%).

Carcinomas showed greater than 50% cells with nuclear staining. Stromaland inflammatory cells were negative throughout.

EXAMPLE 27 Staining of Bronchus with Anti-MCM5 Antibodies

Histological samples of normal and carcinoma bronchus, prepared as forthe paraffin embedded specimens of breast tissue described in Example19, were stained with anti-MCM5 antibody.

Normal cases showed staining of basal proliferating epithelial cellsonly (less than 10%).

Carcinomas showed greater than 50% cells with nuclear staining. Stromaland inflammatory cells were negative throughout.

EXAMPLE 28 Staining of Lymph Nodes, Both Normal and with a Range ofLymphomas, Using Anti-MCM5 Antibody

Both frozen and paraffin embedded histological samples were prepared ofreactive lymph nodes and a range of Hodgkin's and non-Hodgkin'slymphomas as is described for breast tissue in Example 19.

Reactive lymph nodes showed strong staining of cells in the germinalcentre of lymphoid follicles and occasional scattered positive cells inthe parafollicular areas.

Lymphomas showed greater than 50% nuclear staining of malignant lymphoidcells.

EXAMPLE 29 Analysis of Urine Cytology Smears with Anti-MCM5 Antibody

Urine samples were collected from patients with known transitional cellcarcinoma and from normal patients attending urology clinic. Twentymillilitres of urine was spun down at 3,000 g for 10 minutes,supernatant removed and the pellet resuspended in 50 microlitres ofsupernatant. This was smeared onto an APES slide and fixed in alcohol.

The slides were washed in Tris buffered saline (TBS), then permeabilizedin 4 mM sodium deoxycholate for 10 minutes. They were washed with TBSplus 0.025% Triton and blocked with 10% goat serum in TBS for 2 hours.Preabsorbed anti-MCM5 antibody was diluted in TBS containing 0.1% Tritonand 1% BSA and 200 microlitres was added to each slide. Incubation wasovernight at 4° C. in a humidified chamber on an orbital shaker.

The slides were washed in TBS containing 0.025% Triton, followed byincubation in biotinylated goat anti-rabbit secondary antibody (DAKO) at1:500 in TBS containing 1% BSA for 1 hour at room temperature.Endogenous peroxidase was blocked with 0.6% hydrogen peroxide in TBS for10 minutes, followed by wash in TBS. A streptavidin horseradishperoxidase system using the substrate diaminobenzidine was used to stainthe slides. The reaction was stopped by rinsing in water and the slideswere lightly stained with Harris' haematoxylin followed by staining withOrange G and EA50 (PAP stain).

In six cases of transitional cell carcinoma, urine cytology smearsprepared in this way and stained with anti-MCM5 antibodies showed strongstaining of all malignant transitional cells, with unstainedinflammatory and squamous cells in the background. Similar smearsproduced from urine of normal people attending urology clinics showed nostaining of squamous or normal transitional cells.

EXAMPLE 30 DELFIA of Normal Cervical Samples and Cervical Samples fromPatients with Squamous Intraepithelial Lesions

A dissociation enhancement lanthanide fluorescence immunoassay (DELFIA)was established for detection of human MCM5 using two different rabbitpolyclonal anti-MCM5 antisera as described in Example 15 above.

Two samples of normal cervix and two samples of HSIL cervix wereanalysed. The tissue samples were solubilised by hypotonically swellingand douncing followed by salt extraction of DNA bound particles.

The two normal samples gave responses similar to the zero standards.

The two HSIL samples scored positive, indicating that abnormality in acervical sample can be detected using immunoassay.

EXAMPLE 31 Staining of Various Carcinoma Tissues with Anti-MCM5 Antibody

Histological specimens of various carcinomas and leukaemic bone marrowwere stained with anti-MCM5 antibodies, the results being as follows:

Stomach carcinoma showed greater than 50% staining of tumour cells.

Kidney carcinoma showed 30–50% staining of tumour cells.

Ovary carcinoma showed 30–50% staining of tumour cells.

Testis carcinoma showed 30–50% staining of tumour cells.

Bone marrow of acute leukaemia showed greater than 90% staining oftumour cells.

EXAMPLE 32 Staining of Colon Smears

Faecal material was collected from healthy patients and surfaceexfoliated colonocytes were-extracted from the faecal samples by meansof magnetic beads coated with epithelia-specific antibodies, supplied byDynal AS (Oslo, Norway), using the process as described in WO97/09600.

The extracted mixture of magnetic beads and epithelial cells was washedin TBS (Tris-buffered saline) containing 0.025% Triton. After fixationin 4% buffered paraformaldehyde, the cells were washed in TBS and smearswere made from the resulting cell pellet. These were then treated as forsmears from the urine samples.

The PAP stained smears showed a mixture of magnetic beads, somecellulose and cell debris; many columnar epithelial cells from the colonand a few squamous cells from the anal canal were present.

On staining with anti-MCM5 antibodies, similar results are obtained fornormal and abnormal cells as for the bladder or cervix.

EXAMPLE 33 Staining of Bowel Sections of Patients with UlcerativeColitis

Paraffin-embedded sections of bowel from cases of active ulcerativecolitis were stained with antibodies against MCM5.

In all sections tested approximately 50% of the surface epithelial cellsshowed nuclear expression of MCM5 in the inflamed areas. Large numbersof lymphocytes are present in active ulcerative colitis, and these cellsalso showed frequent nuclear expression of MCM5.

Sections of quiescent ulcerative colitis (i.e. with no activeinflammation) were also analysed. In all of these the surface epithelialcells showed no staining for MCM5. Of the small numbers of lymphocytespresent in quiescent ulcerative colitis, only a rare cell showed nuclearstaining for MCM5.

In the paraffin-embedded sections, staining of active and quiescentulcerative colitis was found to be similar for MCM5 and PCNA.

EXAMPLE 34 Staining of Bowel Sections of Patients with Crohn's Disease

Staining of paraffin-embedded section of active Crohn's disease bowelshowed nuclear expression of MCM5 in surface epithelial cells adjacentto regions of ulceration and inflammation. Lymphocytes in the inflammedtissue also showed frequent nuclear expression of MCM5.

Quiescent Crohn's disease bowel tissue was also tested and both thesurface epithelial cells and the small numbers of lymphocytes presentwere negative for MCM5.

Similar findings were obtained for anti-MCM5 antibodies as for anti-PCNAantibodies on the paraffin-embedded cases of active and quiescentCrohn's disease.

A comparison between anti-MCM5 and anti-PCNA staining made on frozensections and paraffin-embedded sections shows that in frozen sectionsstaining with anti-MCM5 antibodies is superior to staining withanti-PCNA antibodies, with more nuclei being stained.

EXAMPLE 35 Staining of Normal and Cancerous Endometrium with Anti-MCM5Antibodies

Frozen and paraffin-embedded sections of normal and cancerousendometrium were stained with anti-MCM5 antibodies.

Good staining was shown in the cancerous endometrium as compared withthe normal tissue, and superior in the frozen sections compared with theparaffin-embedded.

EXAMPLE 36 Staining of Cervical Smear Cell Monolayer (ThinPrep)

After making a smear on an APES slide, the brush/spatula used for takingthe cervical smear was placed in 75% methanol and the remaining cellsremoved by vortexing. The suspension of cells was layered on to 20%sucrose, spun at 1,000 rpm for 2 minutes in a MSE Harrier centrifuge andthe top layer removed and discarded. The remaining layer was spun at3,000 rpm for 5 minutes and the cell pellet resuspended in 200microlitres of water. Fifty microlitres was placed on each slide, thecells allowed to settle and the water removed. The slides were thencarried through as in Example 29 (urine cytology smears) and PAPstained.

In various experiments, results obtained with monolayer smears were thesame as those obtained with conventional smears. Use of monolayerpreparations may be advantageous in that mucus and the majority ofinflammatory cells are removed.

DISCUSSION

The results described herein show that Cdc6 and MCM5 are bothdown-regulated in normal differentiated tissues in vivo and absent fromchromatin in a range of quiescent mammalian cells in culture. Thisprovides some suggestion that these proteins may be of potential valueas cell proliferation markers. Hiraiwa et al. demonstrated that MCM7 canbe immunolocalised in a variety of tumour types such as benign skintumours and malignant tumours of the stomach, pancreas and colon, with asimilar distribution to PCNA. They concluded that MCM7immunolocalization could be applied as an index of cell proliferation intissue sections. However, as noted already above, experts in the fieldof pathology are sceptical that the measurement of cell proliferationrates in tumours by markers such as PCNA & Ki67 will be of any clinicaluse, as there is little direct evidence that such markers are a realimprovement on conventional histological assessments such as grading andstaging, when optimally applied.

The observations reported here show that specific binding moleculesdirected against Cdc6, MCM5 and MCM7 exhibit a much higher degree ofspecificity for potential pre-malignant cells in fresh and frozencervical SILs, than do conventional proliferation markers such as PCNAand Ki67. Anti-Cdc6 and anti-MCM5 are able clearly to discriminate theabnormal cells in LSIL and HSIL from adjacent normal cells, includingendocervical, ectocervical, metaplastic and stromal cells.

In view of this, as described anti-Cdc6 and anti-MCM antibodies wereapplied to cervical smears taken from patients with SILs, and fromdisease free patients. The results were surprising in the strikingdegree of specificity and sensitivity observed for these proteins.Strong nuclear and cytoplasmic staining were observed in both neoplasticcells and HPV-infected koilocytes. Positive immunostaining was alsoidentified in metaplastic squamous cells showing borderlineabnormalities (atypical squamous cells of uncertain significance).However, the remaining mixed population of normal cells in the smearincluding ectocervical cells, endocervical cells, squamous metaplasticcells and inflammatory cells (both lymphocytes and neutrophils) werenegative for Cdc6 and MCM immunostaining.

The sensitivities of anti-MCM5, anti-Cdc6 and anti-MCM7 are much higherthan anti-PCNA when applied to cervical smears and frozen sections, butsimilar patterns of staining are given by such antibodies when appliedto tissue fixed in formalin, paraffin embedded and exposed to antigenretrieval by pressure cooking. Cervical smears and other cytologicalsamples as well as frozen sections are less robust than formalin fixed,paraffin embedded tissue sections and so cannot be subjected to pressurecooking.

The surprising specificity and sensitivity of the Cdc6 and MCMantibodies when applied to cervical smears provides for introduction ofa biochemical/immunocytological approach to mass automated cervicalscreening. Furthermore, these antibodies may help to improve thedetection and classification of LSILs, for which there is at presentmarked intraobserver and interobserver variation in grading, evenamongst experts in the field of cervical cytology. The use of theseantibodies will also help identify HSILs with greater accuracy andobjectivity, thus helping to reduce the high number of false negativeresults, a major problem associated with the present global cervicalscreening programmes.

As noted, the further experimental exemplification of assessment ofbreast tissue, stomach, kidney, ovary, testis and colon, urine samplesand blood samples (both of leukaemia/lymphoma patients and of metastaticsarcoma and carcinoma patients), also tissues of inflammatory boweldisease including ulcerative colitis and Crohn's disease, and faecalsmears indicates the generality of the aspects of the present inventionbeyond cervical screening, though assessment of cervical samples,especially cervical smears, is preferred in various embodiments. Theapplication of biochemical techniques is also demonstrated in additionto cytology.

The results of the blinded trial comparing an embodiment of the presentinvention with assessment of cervical smears using standard PAPsmearing, described in Example 14 above, confirm the exciting utility ofthe invention.

All documents mentioned herein are incorporated by reference.

TABLE I Comparison of anti-Mcm5 antibody test versus conventional Paptest in a blind trial of patients recalled to colposcopy clinicsStandard Pap test result Normal Low grade High grade Anti-Mcm5 antibodytest Presence of  3^(a) 9 17 positive cells Absence of 13 0  0 positivecells ^(a)see text

1. A method of determining the presence or absence of dysplastic or neoplastic cells in a test cytology sample containing cells from an individual, wherein the test sample comprises a specimen selected from the group consisting of a sputum sample, a bronchio-alveolar lavage sample, a urine sample, a breast duct fluid sample, a brushing from the alimentary tract, a cervical cytology sample, a fecal sample, and a urine sample, the method comprising: contacting the test sample with an antibody or antibody fragment directed against Minichromosome Maintenance protein 2 (MCM2 protein); and determining the amount of binding of said antibody or antibody fragment to said test sample; whereby an increase in said amount if detected for the test sample compared with a normal sample is indicative of presence of dysplastic or neoplastic cells in said test sample.
 2. A method according to claim 1 wherein the sample is provided from fluid taken from the individual.
 3. A method according to claim 2 wherein a sample of cells is provided from said fluid.
 4. A method according to claim 2 wherein the fluid is urine.
 5. A method according to claim 1 wherein a population of individuals is screened.
 6. A method according to claim 1 wherein the sample is a cervical smear.
 7. A method according to claim 6 wherein a population of individuals is screened.
 8. A method according to claim 2 wherein a population of individuals is screened.
 9. A method according to claim 4 wherein a population of individuals is screened. 